Fig. 5

HuR regulates telomerase activity via TERC methylation. a Forty-eight hour after transfecting HeLa cells with a HuR siRNA (black) or a control siRNA (blank), RNA was isolated and used for bisulfite RNA sequencing analysis to measure the methylation of C106 and C323. b Forty-eight hour after transfecting HeLa cells with a vector expressing TERC variants or an empty vector (WT), RNA was isolated and used for bisulfite RNA sequencing analysis to measure C106 methylation in different variants (bearing point mutations were used for analysis). c U2OS cells were co-transfected with a vector expressing TERC variants C106G or U40A, U100A, or U40A + U100A, or C106G. Forty-eight hour later, TRAP assays were performed to determine the telomerase activity. d U2OS cells were co-transfected with a vector expressing flag-TERT together with a vector expressing TERC or its variant bearing C106G. UV crosslinking followed by RNP IP assays were performed to evaluate the association of flag-TERT with TERC and the variant bearing C106G. e HeLa cells were transfected with a vector expressing flag-hTERT or an empty vector (Input). Forty-eight hours later, lysates were prepared and subjected to UV crosslinking followed by IP assays using an anti-flag antibody. RNA prepared from the IP materials was further used for bisulfite RNA sequencing analysis to assess the methylation of C106. Data in c–d were normalized against the levels of TERC and its variants. Data in a, b, c, d, and e represent the means ± SD from three independent experiments; significance is analyzed by Student’s t-test (**p < 0.01)